Abstract

Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve.

Authors

Figure 6

Cells infiltrate into the ONHs of glaucoma-prone eyes prior to glaucomatous damage but were not detected in radiation-treated eyes.

(A–F) IBA1+ cell numbers were assessed from the ONH to the myelinated region (anterior to white line) of 10.5-month-old DBA/2J, Rad-D2, and D2-Gpnmb+ control eyes by immunofluorescence. (E) An example of the counted IBA1+ cells is shown. The number of IBA1+ cells increased in untreated eyes compared with that in controls but there are significantly fewer in Rad-D2 eyes (P = 0.0002). (G–K) Cell infiltration was assessed using CFDA (injected into the spleens; see Methods). CFDA+ cells had entered the optic nerves of untreated DBA/2J eyes (n = 8) but not Rad-D2 eyes (n = 6) or control eyes (n = 6). Fluorescent cells must have taken up CFDA in the spleen. H is a merged view of J (fluorescent) and K (DIC). Arrows indicate location of CFDA+ cells. (L) Flow cytometric analysis revealed that CD45hiCD11b+ monocyte numbers increased in the ONHs of untreated 10.5-month-old DBA/2J eyes compared with those in D2-Gpnmb+ controls and radiation-treated eyes (P = 0.01). CD45hiCD11b+ monocyte numbers did not increase in radiation-treated eyes compared with D2-Gpnmb+ controls (P = 0.96). CD45hi is a marker of blood-derived infiltrating cells. One-third of the CD45hiCD11b+ cells were also positive for the proinflammatory marker Ly6c+. (M) Flow cytometric analysis also shows that the CD45hi cells that infiltrate into DBA/2J eyes are CD11b+CD11c+ double-positive monocytes. This class of cell is completely absent in radiation-treated eyes (P = 0.0007, compared with untreated eyes). Scale bars: 50 μm (A–C and G–I); 20 μm (D and E and J and K).